Synthesis, photophysical properties, and application of o - And p -amino green fluorescence protein synthetic chromophores

Article abstract of DOI:10.1021/jo302050y

The o- and p-amino green-fluorescence-protein synthetic chromophores (GFPSCs) were synthesized from the corresponding o- and p-nitro protecting group. Among the four protecting groups of the o-amino group, the o-nitro protecting group is the only choice to synthesize the o-amino GFPSCs. The first singlet excited states of o- and p-amino GFPSCs carry significant charge-transfer character through the mechanism of photoinduced charge transfer (PCT). The o-amino GFPSCs can serve as wavelength-ratiometric fluorescence sensors that selectively recognize Cr³⁺ in aqueous medium through a PCT mechanism.

Full text of DOI:10.1021/jo302050y

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Article
Synthesis, Photophysical Properties and Application of o- and
p-Amino Green Fluorescence Protein Synthetic Chromophores
Yi-Hui Chen, Wei-Jen Lo, and Kuangsen Sung
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/jo302050y • Publication Date (Web): 29 Nov 2012
Downloaded from http://pubs.acs.org on December 11, 2012
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Synthesis, Photophysical Properties and Application of o- and
p-Amino Green Fluorescence Protein Synthetic Chromophores
YiꢀHui Chen, WeiꢀJen Lo and Kuangsen Sung*
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Department of Chemistry, National Cheng Kung University, Tainan, Taiwan
kssung@mail.ncku.edu.tw
ABSTRACT: The oꢀ and pꢀamino greenꢀfluorescenceꢀprotein synthetic
chromophores (GFPSCs) were synthesized from the corresponding oꢀ and pꢀnitro
protecting group. Among the four protecting groups of the oꢀamino group, the oꢀnitro
protecting group is the only choice to synthesize the oꢀamino GFPSCs. The first
singlet excited states of oꢀ and pꢀamino GFPSCs carry significant chargeꢀtransfer
character through the mechanism of photoinduced charge transfer (PCT). The
oꢀamino GFPSCs can serve as wavelengthꢀratiometric fluorescence sensors that
3+
selectively recognize Cr in aqueous medium through PCT mechanism.
INTRODUCTION
The green fluorescent protein (GFP) and its family have been emerging as
1
powerful probes for intracellular dynamics. The GFP absorbs at 395 nm (neutral
form) with a weaker absorption at 475 nm (anion form), and emits green light at 508
nm that is anion emission caused by intermolecular excitedꢀstate protonꢀtransfer
1
(
ESPT). When the GFP is stripped of the protein environment, the central free
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chromophore of pꢀhydroxybenzylidenedimethylimidazolone (pꢀHBDI) does not
2b
display a tautomer emission through intermolecular ESPT in all solvents and its
fluorescence quantum yield significantly decreases because of photoisomerization
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decay or internal conversion. On the other hand, oꢀHBDI shows a tautomer emission
2a,c
at 600 nm that involves excitedꢀstate intramolecular proton transfer (ESIPT). We
wondered how replacement of hydroxyl group of GFP chromophore with an amino
group might alter its photophysics and what its potential application might be.
3+
Trivalent chromium, Cr , is an important trace element in human biological
4a,b
system because it involves several biochemical processes.
Diabetes and
3
+
4c
cardiovascular diseases are associated with Cr deficiency. Cellular structures can
3+ 4d
be negatively affected by exposure to high level of Cr . Hence, detection of trace
3
+
Cr is in high demand. Due to simplicity, high sensitivity and instantaneous response,
fluorescence detection of metal ions usually provide great advantages over other
5
a,b
5cꢀi
detection techniques. There are some photoluminescent ratiometric
or turnꢀon
3
+
sensors that were developed for recognition of Cr through the following
5a
chromophore/mechanism: heavy metal complex/PET, rodamine/ringꢀopening(or
5b,fꢀh
5c
5d
FRET),
chalcogeno
podand/conformation
change,
BODIPY/PET,
5e
5i
dansyl/PET, and 4ꢀaminophthalimide/PET . In spite of these advances, the sensors
still suffer from some inadequacies like selectivity, reversibility, or use in aqueous
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medium. In this paper, we used oꢀamino GFPSC as a chromophore and photoinduced
charge transfer (PCT) as a sensing mechanism to develop wavelengthꢀratiometric
3
+
fluorescence sensors that selectively recognize Cr and can be used in aqueous
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medium.
COMPUTATIONAL DETAILS
6
All the calculations reported here were performed with the Gaussian03 program.
Geometry optimization of oꢀ4, oꢀ7 and pꢀ7 was carried out at B3LYP/6ꢀ31+G*
without any symmetry restriction. After the geometry optimization was performed,
analytical vibration frequencies were calculated at the same level to determine the
nature of the located stationary point. Timeꢀdependent density functional theory
(TDDFT) was used to calculate the vertical electronic transitions of the first 5 states
of oꢀ4, oꢀ7 and pꢀ7 in the gas phase and the electronꢀdensity surfaces of their
molecular orbital at the level of B3LYPꢀTD/ccꢀPVDZ//B3LYP/6ꢀ31+G*.
RESULT AND DISCUSSION
Synthesis. One of the potential problems for the preparation of oꢀ4 is that the
oꢀamino group as a good nucleophile may undergo intramolecular cyclization during
syntheses. To solve the problem, our strategy is to use four kinds of protecting groups
for the oꢀamino group, and its retroꢀsynthesis is outlined in Scheme 1.
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Because 5ꢀoxazolone is susceptible to nucleophilic attack, we recover the
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oꢀamino group after the 5ꢀoxazolone is converted to 5ꢀimidazolone. In this
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retroꢀsynthesis, oꢀ2a has been done by Gucky through the step 1. However, we tried
this reaction many times with various conditions, and we always obtained 5, instead
of oꢀ2a. (Scheme 2) The oꢀacetamido group undergoes intramolecular cyclization and
fails to serve as a protecting group of the oꢀamino group.
0
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Scheme 1. Retroꢀsynthesis of oꢀAmino GFPSC (oꢀ4)
O
O
Step 3
N
N
N
N
NH2
R
oꢀ3
o-4
O
O
O
HN
H
Step 2
Step 1
O
+
N
R
O
R
oꢀ2
oꢀ1
NHꢀ
OH
a: R = CH C(O)NHꢀ
c: R = pꢀtolylꢀSO
d: R = O Nꢀ
2
3
b: R = PhC(O)NHꢀ
2
Scheme 2. Reaction of oꢀ1a with NaOAc/Ac O in the Presence of NꢀAcetylglycine
2
under Reflux
O
O
O
O
2
Ac O
N
NaOAc
NH
N
H
oꢀ1a
oꢀ2a
O
O
NaOAc
H
N
Ac O
2
OH
N
H
O
O
N
O
5
H
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When oꢀ1a reacted with NaOAc/Ac
2
O under reflux in the absence of
Nꢀacetylglycine, 6 was generated and the oꢀamido group also got involved in the
intramolecular cyclization, indicating that αꢀcarbanion of the oꢀacetamido group
serves as a nucleophile for the cyclization reaction. (Scheme 3) Thus, we used the
oꢀbenzamido group to replace the oꢀacetamido group as a protecting group of the
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2
oꢀamino group. Then, oꢀ1b reacted with NaOAc/Ac O under reflux in the presence of
Nꢀacetylglycine, and 5 was generated again. (Scheme 4) The oꢀbenzamido group also
got involved in the intramolecular cyclization. The strange thing is that the benzoyl
group was removed during the reaction. Hence, we replaced the oꢀbenzamido group
with oꢀtosylamido group as a protecting group of the oꢀamino group. When oꢀ1c
2
reacted with NaOAc/Ac O under reflux in the presence of Nꢀacetylglycine, 5 was
generated again and the tosyl group was also removed during the intramolecular
cyclization. It is unusual that the acety, benzoyl, and tosyl groups are removed from
the oꢀamino group during the intramolecular cyclization.
Scheme 3. Reaction of oꢀ1a with NaOAc/Ac O in the Abscence of NꢀAcetylglycine
2
under Reflux
O
O
NaOAc
Ac O
N
O
N
2
H
H
6
oꢀ1a
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Scheme 4. Reaction of oꢀ1a, oꢀ1b or oꢀ1c with NaOAc/Ac
2
O in the Presence of
NꢀAcetylglycine under Reflux
O
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H
N
NaOAc
Ac O
2
R
O
N
N
O
H
5
H
oꢀ1a, 1b or 1c
O
oꢀ1a: R = CH C(O)ꢀ
3
OH
oꢀ1b: R = PhC(O)ꢀ
N
H
oꢀ1c: R = pꢀtolylꢀSO2ꢀ
O
Scheme 5. Synthesis of oꢀ and pꢀAmino GFPSCs
O
O
O
OH
H
N
1. MeNH /H O
2
2
O
H
O
N
NO2
2. pyridine
NO2
Ac O/NaOAc
2
oꢀ1d
pꢀ1d
oꢀ2d
pꢀ2d
O
O
O
2.2 eq.
CH3I
Pd/C
N
N
N
N
N
N
NMe
2
NO2
H / MeOH
2
NH2
oꢀ7
oꢀ4
pꢀ4
oꢀ3d
pꢀ3d
Because oꢀacetamido, oꢀbenzamido and oꢀtosylamido groups fail to serve as
protecting groups for the oꢀamino group in the preparation of oꢀ4, we used the nitro
group as a protecting group of the oꢀamino group. When oꢀ1d reacted with
NaOAc/Ac O under reflux in the presence of Nꢀacetylglycine, oꢀ2d was generated
2
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and no intramolecular cyclization occurred. (Scheme 5) To convert 5ꢀoxazolone to
5
ꢀimidazolone, oꢀ2d was treated with methylamine, followed by reflux in pyridine, to
get oꢀ3d. Then, the oꢀamino group is recovered by Pdꢀcatalyzed hydrogenation of
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2 2
oꢀ3d in MeOH. We tried to use SnCl or Fe to reduce the NO group of oꢀ3d, but
these reactions led to very complicated products. The oꢀN,Nꢀdimethylamino analogue
oꢀ7 was produced by treating oꢀ4 with 2.2 equivalent of methyl iodide.
Photophysics. The lowestꢀenergy electronic absorption bands of oꢀ4, oꢀ7, pꢀ4
3
ꢀ1
ꢀ1
3
and pꢀ7 in acetonitrile are located at 422 (ε = 5.3 ×10 M cm ), 396 (ε = 6.7 ×10
ꢀ1
ꢀ1
4
ꢀ1
ꢀ1
4
ꢀ1
ꢀ1
M cm ), 394 (ε = 2.7 ×10 M cm ) and 422 (ε = 2.5 ×10 M cm ) nm, respectively.
(Table 1) To understand whether the lowestꢀenergy electronic absorption band of oꢀ4,
oꢀ7, pꢀ4 and pꢀ7 involve a chargeꢀtransfer absorption, we use nitrobenzene as a
reference and do calculations on its chargeꢀtransfer absorption by TDDFT. It is known
that an observed chargeꢀtransfer absorption of nitrobenzene is located at 252 nm with
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f=0.17. Its calculated chargeꢀtransfer absorption, which is contributed by
0
.64[HOMOꢀ1ꢀLUMO]ꢀ0.13[HOMOꢀLUMO+1], with oscillator strength (f) is
2
55 nm (f=0.1963) at the B3LYPꢀTD/ccꢀPVTZ//B3LYP/6ꢀ31+G* level, and 249 nm
(f=0.1902) at the B3LYPꢀTD/ccꢀPVDZ//B3LYP/6ꢀ31+G* level, both of which are in
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good agreement with the experimental result. (Table 2) Transition electric dipole
moment calculated by TDDFT has been used to see if an electronic transition involves
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a charge transfer and to see the direction of electron transfer.
At the
B3LYPꢀTD/ccꢀPVDZ//B3LYP/ 6ꢀ31+G* level, the transition electric dipole moment
for the chargeꢀtransfer absorption of nitrobenzene at 249 nm is 1.248 a.u. (3.2 Debye)
along xꢀaxis, indicating that a substantial electron transfer has occurred from the
benzene ring to the nitro group. (Fig. 1) This is consistent with the experimental
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result.
Table 1. Photophysical Properties of oꢀ and pꢀAmino GFPSCs at Room Temp.
oꢀ4
oꢀ7
pꢀ7

a
a
a
a
a
a
Solvent abs

em
abs
em
abs
em
C
6
H
12
427 451, 465(2)
422 452, 489(4)
395
396
395
396
398
398
346
472(10)
535(6)
519(14)
536(8)
552(5)
522(3)
426(3)
405
428
419
422
434
435
446
437(0.1)
482(0.9)
452(0.4)
483(0.3)
490(0.2)
492(0.3)
CH
2
Cl
2
THF
430
513(9)
3
CH CN 422 453, 524(4)
DMSO
436
530(7)
3
CH OH 424 454, 520(2)
H
2
O
390 452, 488(1)
520(0.2)
a.
ꢀ3
Unit: nm; emission wavelength with fluorescence quantum yield (φ
f
×10 ) inside
bracket
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Table 2. Calculated and Observed Electronic Absorption, Oscillator Strength (f), and
Transition Electric Dipole Moment (ꢀtr) of Nitrobenzene, oꢀ4, oꢀ7 and pꢀ7
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Compound
λabs(nm)
f
ꢀtr (a.u.)
a
calcd / level exptl calcd exptl
x
1.2837
1.248
ꢀ1.9
y
z
b
f
f
f
Nitrobenzene
Nitrobenzene
oꢀ4
255 /A
252 0.1963 0.17
0
0
b
f
249 /B
252 0.1902 0.17
0
0
c
g
g
g
415 /B
427 0.2894 0.11
ꢀ0.59
0.05
0.11
0
d
g
oꢀ7
410 /B
395 0.2153 0.17
1.7
ꢀ0.08
0
e
g
g
pꢀ7
382 /B
405 0.9418 0.52
ꢀ3.4
a.
A: B3LYPꢀTD/ccꢀPVTZ//B3LYP/ 6ꢀ31+G*; B: B3LYPꢀTD/ccꢀPVDZ//B3LYP/
ꢀ31+G*.
This is the chargeꢀtransfer absorption contributed by 0.64[HOMOꢀ1ꢀLUMO]
0.13[HOMOꢀLUMO+1].
6
b.
c.
d.
e.
9
ꢀ
This is the lowestꢀenergy electronic absorption contributed by 0.61[HOMOꢀ
LUMO]ꢀ0.18[HOMOꢀ1ꢀLUMO].
This is the lowestꢀenergy electronic absorption contributed by 0.58[HOMOꢀ
LUMO]ꢀ0.33[HOMOꢀ1ꢀLUMO].
This is the lowestꢀenergy electronic absorption contributed by 0.61[HOMOꢀ
LUMO]
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f.
Ref. 9
g.
In cyclohexane
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Figure 1. Electronꢀdensity surfaces for HOMOꢀ1, HOMO, LUMO and LUMO+1 of
nitrobenzene
We do calculations on the electronic transitions of oꢀ4, oꢀ7 and pꢀ7 at the
B3LYPꢀTD/ccꢀPVDZ//B3LYP/6ꢀ31+G* level to understand whether their
lowestꢀenergy electronic absorption bands involve a chargeꢀtransfer transition. The
calculated lowestꢀenergy singlet electronic transition of oꢀ4 is contributed by
0
.61[HOMOꢀLUMO]ꢀ0.18[HOMOꢀ1ꢀLUMO] and located at 415 nm with an
oscillator strength (f) of 0.2894 and a transition electric dipole moment of ꢀ1.9 a.u.
(ꢀ4.87 Debye) along the x axis and ꢀ0.59 a.u (ꢀ1.51 Debye) along the y axis, which is
in good agreement with the experimental data (427 nm in cyclohexane with ε = 5167
ꢀ1
ꢀ1
M cm , which corresponds to oscillator strength (f) of 0.11 according to the
11
ꢀ9
ꢀ9
equation of f = 4.3×10 ∫εdν = 4.3×10 ×εmax×ꢁν1/2). The magnitude of this
transition electric dipole moment is greater than those of the chargeꢀtransfer
transitions of nitrobenzene and peridinin (3.2 Debye for nitrobenzene and 3.4 Debye
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1
0b
for peridinin ). Hence, we suggest that the lowestꢀenergy electronic absorption of
oꢀ4 involves a chargeꢀtransfer transition. The negative sign of this transition electric
dipole moment of oꢀ4 indicates that a substantial electron transfer has occurred from
the imidazolone moiety to the aniline moiety during the excitation. (Fig. 2)
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45
46
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48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. Electronꢀdensity surfaces for HOMOꢀ1, HOMO and LUMO of oꢀ4
The calculated lowestꢀenergy singlet electronic transition of oꢀ7 is contributed by
0.58[HOMOꢀLUMO]ꢀ0.33[HOMOꢀ1ꢀLUMO] and located at 410 nm with an
oscillator strength (f) of 0.2153 and a transition electric dipole moment of 1.7 a.u.
(
4.36 Debye) along the xꢀaxis, 0.05 a.u (0.13 Debye) along the yꢀaxis, and ꢀ0.08 a.u
ꢀ0.21 Debye) along the zꢀaxis, indicating that a substantial electron transfer has
(
occurred from the imidazolone moiety to the aniline moiety during the excitation. (Fig.
) According to the magnitude and sign of this transition electric dipole moment, we
3
suggest that the lowestꢀenergy electronic absorption of oꢀ7 involves a chargeꢀtransfer
transition. This result is in good agreement with the experimental data (395 nm in
ꢀ1
ꢀ1
cyclohexane with ε = 7940 M cm , which corresponds to oscillator strength (f) of
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9
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0.17).
0
1
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8
9
0
1
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9
0
1
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Electronꢀdensity surfaces for HOMOꢀ1, HOMO and LUMO of oꢀ7
The calculated lowestꢀenergy singlet electronic transition of pꢀ7 is contributed by
0
.61[HOMOꢀLUMO] and located at 382 nm with an oscillator strength (f) of 0.9418
and a transition electric dipole moment of ꢀ3.4 a.u. (ꢀ8.72 Debye) along the xꢀaxis and
.11 a.u. (0.28 Debye) along the yꢀaxis, indicating that a substantial electron transfer
0
has occurred from the aniline moiety to the imidazolone moiety during the excitation.
(Fig. 4) This is opposite to those of oꢀ4 and oꢀ7, indicating that the orientation of
aniline has a significant influence on the direction of electron transfer in the
chargeꢀtransfer transition. According to the magnitude and sign of this transition
electric dipole moment, we suggest that the lowestꢀenergy electronic absorption of pꢀ7
involves a chargeꢀtransfer transition. This result is in good agreement with the
ꢀ1
ꢀ1
experimental data (405 nm in cyclohexane with ε = 23966 M cm , which
corresponds to oscillator strength (f) of 0.52).
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51
52
53
54
55
56
57
58
59
60
Figure 4. Electronꢀdensity surfaces for HOMO and LUMO of pꢀ7
The lowestꢀenergy electronic absorption of pꢀ7 is redꢀshifted in the polar
solvents. This is likely because the chargeꢀtransfer excitedꢀstate is more polar than the
groundꢀstate and the dipoleꢀdipole interactions with solvent molecules lower the
11
energy of the excited state more than that of the ground state. This redꢀshift trend is
not found for the lowestꢀenergy electronic absorption of oꢀ4 and oꢀ7 in the polar
solvents, even though they also involve a chargeꢀtransfer transition. This is likely
because steric hindrance between the aniline and the imidazolone moieties play a
more important role. The lowestꢀenergy electronic absorption bands of oꢀ4 and oꢀ7 in
water are significantly blueꢀshift relative to those in other solvents, and this is likely
attributed to the increasing twist angle between the aniline and the imidazolone
moieties, which is caused by the strong solvation in water through intermolecular
hydrogenꢀbonding. In addition, the significant blueꢀshift for the lowestꢀenergy
electronic absorption band of oꢀ7 relative to that of oꢀ4 is also attributed to the
increasing twist angle between the aniline and the imidazolone moieties.
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The oꢀ4 displays dual fluorescence in acetonitrile: one at 453 nm that is not
sensitive to solvent polarity and is ascribed to radiation relaxation from the locally
1
2
excited (LE) state, and the other at 524 nm that is sensitive to solvent polarity. (Fig.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
8
9
0
1
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0
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8
9
0
1
2
3
4
5
6
7
8
9
0
5
) The pꢀ4 is nonꢀfluorescent while the emissions at 536 nm of oꢀ7 and at 483 nm of
pꢀ7 in acetonitrile are also sensitive to solvent polarity. (Fig. 6 and 7) According to the
13
LippertꢀMataga equation (Eq. 1), solvatochromic Stokes shifts (ꢁν) of the
longestꢀwavelength emission of oꢀ4, oꢀ7 and pꢀ7 were plotted as a function of the
solvent polarity parameter ꢁf. (Fig. 8) These three LippertꢀMataga plots are linear and
their slopes (ꢁν/ꢁf) in combination with the radius of the cavity (a) can be used to
calculate the dipoleꢀmoment changes (ꢁꢀ) from the groundꢀstates to the first singlet
excitedꢀstates of oꢀ4, oꢀ7 and pꢀ7, which were calculated to be 10.8, 10.9 and 7.9
Debye, respectively, indicating that the first singlet excitedꢀstates of oꢀ4, oꢀ7 and pꢀ7
bear significant chargeꢀtransfer character. Hence, the longestꢀwavelength emissions of
oꢀ4, oꢀ7 and pꢀ7 are ascribed to radiation relaxation from the twisted internal
12
chargeꢀtransfer (TICT) states. The first singlet excitedꢀstate of pꢀ7 shows less
chargeꢀtransfer character than those of oꢀ4 and oꢀ7, indicating that a longer distance
between electronꢀdonor and electronꢀacceptor reduces chargeꢀtransfer degree. This is
1
4
consistent with the RehmꢀWeller equation. In contrast to oꢀ7 and pꢀ7, the dual
fluorescence of oꢀ4 is found in less polar solvents of dichloromethane and
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cyclohexane and protic solvents of methanol and water. It is likely that the
intramolecular hydrogen bonding through NꢀHꢁꢁꢁN or a series of protic solvent
molecules makes the emission from the LE state comparable relative to that from the
TICT state.
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2
3
ꢁ
ν = νabs ꢀ νem = [2/(4πε hc)][(ꢁ
ꢀ
) /a ](ꢁf) + constant
(Eq. 1)
0
2
2
,
where ꢁf = [(εꢀ1)/(2ε+1)]ꢀ[(n ꢀ1)/(2n +1)], a: the radius of the cavity in which the
solute resides. (a = 4.97, 5.32, 5.33 Ǻ for oꢀ4, oꢀ7 and pꢀ7 at B3LYP/6ꢀ31+G* level)
Figure 5. The emission spectra of oꢀ4 in various solvents
Figure 6. The emission spectra of oꢀ7 in various solvents
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2
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 7. The emission spectra of pꢀ7 in various solvents
Figure 8. The LippertꢀMataga plots of oꢀ4 (blue dot), oꢀ7 (red dot) and pꢀ7 (green
dot)
Application. Because the firstꢀsinglet excitedꢀstates of oꢀ4 and oꢀ7 carry
significant chargeꢀtransfer character through the PCT mechanism, they have potential
to become wavelengthꢀratiometric fluorescence sensors, which permit ratiometric
measurements of fluorescence to be made and allow the determination of cation
concentration independently of various parameters like sensor concentration and
15
incidentꢀlight intensity.
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Absorption and fluorescence titrations of oꢀ4 or oꢀ7 with various cations of group
+
+
2+
2+
2+
2+
2+
2+
+
I (Na , K ), group II (Mg , Ca ), and heavy metals (Pb , Zn , Ni , Co , Ag ,
2
+
3+
2+
3+
3
Fe , Fe , Cu , and Cr ) were performed in aqueous CH CN (6:4) at 25℃. What we
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60
3
+
found was that both oꢀ4 and oꢀ7 can selectively recognize Cr .
3
+
We synthesized the 1:3 Cr /oꢀ4 complex by mixing the concentrated oꢀ4 with
0
.33 equivalent of concentrated Cr(NO3)3(aq) in methanol for 30 min at room
temperature until oꢀ4 was completely consumed. We monitored this reaction by
3+
thinꢀlayer chromatography (TLC). The emission maximum of the 1:3 Cr /oꢀ4
complex in aqueous CH CN (6:4) is at 450 nm, which is blueꢀshifted relative to that
3
of oꢀ4 (550 nm). (Figure 9) When the concentrated oꢀ4 was treated with 1.0
equivalent of concentrated Cr(NO3)3(aq), the emission maximum of the product was
blueꢀshifted from 550 to 400 and 380 nm. The dual emission maxima were assigned
3+
to the 1:2 and 1:1 Cr /oꢀ4 complexes. When oꢀ4 was treated with 230 equivalent of
3
+
Cr , its emission maximum at 380 nm increased but the one at 400 nm faded away.
3
+
Hence, the product was supposed to be the 1:1 Cr /oꢀ4 complex. Similar phenomena
were also found in the case of oꢀ7.
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1
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8
9
0
Figure 9. Emission spectra (λex=300 nm) in aqueous CH
produced by mixing concentrated oꢀ4 with (a) 0, (b) 0.33, (c) 1.0, and (d) 230
equivalent of concentrated Cr(NO
3
CN (6:4) for the products
3
)3(aq) in methanol
3+
To see if ethylenediamine competes with oꢀ4 in the recognition of Cr , the 1:3
3
+
Cr /oꢀ4 complex was titrated with excess of ethylenediamine. (Figure 10) During the
3
+
fluorescence titration, the emission maximum was blueꢀshifted from the 1:3 Cr /oꢀ4
3+
complex to the 1:1 Cr /oꢀ4 complex. This indicates that ethylenediamine ligand
3+
easily displaces the first two bidentate ligands of oꢀ4 in the 1:3 Cr /oꢀ4 complex but
it does not displace the last bidentate ligand of oꢀ4. This implies that the binding
3+
3+
affinity between Cr and oꢀ4 is much stronger than that between Cr and
ethylenediamine. The reason why the first two bidentate ligands of oꢀ4 in the 1:3
3
+
Cr /oꢀ4 complex were easily displaced by ethylenediamine is likely because the
3+
bulky oꢀ4 causes significant steric tension in the 1:3 Cr /oꢀ4 complex. Thus, we used
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3
+
3+
the 1:1 Cr /oꢀ4 (or oꢀ7) complex as the final product in the recognition of Cr with
3+
oꢀ4 or oꢀ7, and make sure that [oꢀ4] or [oꢀ7] is less than [Cr ].
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60
3
+
ꢀ4
Figure 10. Fluorescence titration (λex=300 nm) of the 1:3 Cr /oꢀ4 complex (1×10
M) with 0, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2000 equivalent of
ethylenediamine in CH CN
3
3
+
In the UVꢀvis titration, upon addition of Cr , the absorbance at 396 nm of oꢀ7
decreases gradually while the absorbance at 350 nm increases simultaneously,
3+
indicating that Cr chelation reduces the resonance contribution from the lone pair of
electrons on N. (Figure 11) The similar phenomena also happen in the UVꢀvis
titration of oꢀ4. There are isosbestic points that appear in the UVꢀvis titrations of oꢀ4
3+
and oꢀ7 with Cr , indicating that only one complex was formed in each titration
system. According to what we mentioned in Figure 9, we suggest that the only formed
3
+
3+
complex would be the 1:1 Cr /oꢀ4 or Cr /oꢀ7 complex.
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1
2
3
4
5
6
7
8
9
0
ꢀ
4
Figure 11. UVꢀvis titration of (A) oꢀ4 (1×10 M) with 0, 25.6, 51.2, 76.8, 102, 128
ꢀ4
ꢀ5
3
×10 M of Cr(NO )3(aq) and (B) oꢀ7 (1.8×10 M) with 0, 6.7, 13, 20, 27, 33, 40, 47,
ꢀ5
3 3
53, 60 ×10 M of Cr(NO )3(aq) in aqueous CH CN (6:4)
3
+
For the fluorescence titration of oꢀ7 with Cr , the emission at 545 nm decreases
while a new emission at 422 nm increases with 6.7ꢀfold fluorescence enhancement for
3
+
3
3.3 equiv of Cr . (Fig. 12) The blueꢀshift in the emission spectrum is due to the
3+
diminishment of PCT, which is caused by the bidentate chelation of Cr with oꢀ7.
The fluorescence intensity ratio (F422 nm/F545 nm) increases with the concentration of
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+
3+
Cr , and 33.3 equiv of Cr is required to reach its saturation. According to Tsien’s
16
3+
d
method, the dissociation constant (K ) of the 1:1 Cr /oꢀ7 complex was estimated to
3+
be 94 ꢀM. For the fluorescence titration of oꢀ4 with Cr , the emission at 550 nm
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decreases while a new emission at 380 nm increases with 3.7ꢀfold fluorescence
3+
enhancement for 230 equiv of Cr . (Fig. 12) The fluorescence intensity ratio (F380
3+
nm/F550 nm) of oꢀ4 increases with the concentration of Cr until its saturation is reached
3+
3+
at 230 equiv of Cr . The dissociation constant (K
d
) of the 1:1 Cr /oꢀ4 complex was
estimated to be 1.4 mM, which is around 10 times bigger than that of the 1:1
3
+
Cr /oꢀ7 complex.
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0
1
2
3
4
5
6
7
8
9
0
ꢀ
4
Figure 12. Fluorescence titration (λex=300 nm) of (A) oꢀ4 (1×10 M) with 0, 25.6,
ꢀ4
ꢀ5
5
3
1.2, 76.8, 102, 128, 154, 179, 205, 230 ×10 M of Cr(NO )3(aq) and (B) oꢀ7 (1.8×10
ꢀ5
M) with 0, 6.7, 13, 20, 27, 33, 40, 47, 53, 60 ×10 M of Cr(NO
3
)3(aq) in aqueous
3
CH CN (6:4)
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CONCLUSION
In the preparation of oꢀ4, the oꢀacetamido, oꢀbenzamido and oꢀtosylamido groups
fail to serve as protecting groups for the oꢀamino group. Not only do they get involved
in the intramolecular cyclization, but the acetyl, benzoyl and tosyl groups are also
removed during the cyclization. The oꢀnitro protecting group is the only choice to
synthesize the oꢀamino GFPSCs.
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The lowestꢀenergy electronic absorption of pꢀ7 is redꢀshifted in the polar
solvents while this redꢀshift trend is not found for the lowestꢀenergy electronic
absorption of oꢀ4 and oꢀ7 in the polar solvents because the steric hindrance between
the aniline and the imidazolone moieties play a more important role for the latter. Due
to the increasing twist angle between the aniline and the imidazolone moieties by
strong solvation in water, the lowestꢀenergy electronic absorption bands of oꢀ4 and
oꢀ7 in water are significantly blueꢀshift relative to those in other solvents. The first
singlet excited states of oꢀ4, oꢀ7 and pꢀ7 carry significant chargeꢀtransfer character
through the mechanism of PCT.
Both oꢀ4 and oꢀ7 can serve as wavelengthꢀratiometric fluorescence sensors that
3+
selectively recognize Cr and can be used in aqueous medium.
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EXPERIMENTAL SECTION
1
7a
17b
17c
17d
17e
17f
Materials and Methods. The oꢀ1a, oꢀ1b, oꢀ1c, oꢀ2d, pꢀ4 and pꢀ7
0
1
2
3
4
5
6
7
8
9
0
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2
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9
0
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are known and prepared according to the literature methods. High resolution mass
(HRMS) measurements were obtained with magneticꢀelectric sector mass analyzer.
1,2-Dimethyl-4-(2-nitrobenzylidene)-1H-imidazol-5(4H)-one (
(3.48 g, 15 mmol) in 45 mL of THF was slowly added 40 wt. % methylamine (1.164g,
5mmol). The mixture was stirred in a nitrogen atmosphere at room temperature for
o-3d). To oꢀ2d
1
around 20 min until the reaction was complete. Then, the solution was concentrated
by a rotary evaporator, and pyridine (7.5 mL) was added to the residue. The mixture
was stirred in a nitrogen atmosphere under reflux for 5 h. After reaction, the solution
was cooled down and some ice was added to the solution. The solution was stirred
until most of solid precipitated. The crude product was purified by column
chromatography with hexane/ethyl acetate (1:1) as a mobile phase to get oꢀ3d, and the
1
yield was 55%. HꢀNMR (CDCl
3
, 300MHz): 2.37 (s, 3H, CH
3
), 3.20 (s, 3H, CH
3
),
7.48 (t, 1H, PhH), 7.50 (s, 1H, CH), 7.66 (t, 1H, PhH), 8.00 (d, 1H, PhH), 8.46 (d, 1H,
13
3
PhH) ; CꢀNMR (CDCl , 75MHz): 15.7, 26.7, 121.1, 124.6, 128.7, 129.7, 132.9,
1
11 3 3
33.6, 141.0, 149.2, 165.3, 169.9; HRMS (EI) m/z calcd for 245.08, C12H N O ,
found 245.0799
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Cis-4-(2-Aminobenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one (o-4). To
oꢀ3d (1.9 g, 7.75 mmol) in 25 mL of MeOH was added 380 mg of 5% Pd/C. The
mixture was stirred in a hydrogen atmosphere (1 atm) at room temperature for 2—3 h
until the reaction was complete. After filtration, the filtrate was concentrated by a
rotary evaporator. The crude product was purified by column chromatography with
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hexane/ethyl acetate (1:1) as a mobile phase to get oꢀ4, and the yield was 70%.
1
3 3 3
HꢀNMR (CDCl , 300MHz) δ 2.32 (s, 3H, CH ), 3.18 (s, 3H, CH ), 6.25 (s. 1H, NH),
6
7
.62ꢀ6.66 (m, 2H, PhH), 7.15 (t, J = 7.3 Hz, 1H, PhH), 7.21 (s, 1H, CH), 7.49 (d, J =
1
3
.9 Hz, 1H, PhH); CꢀNMR (CDCl
3
, 75MHz) δ 15.5, 26.7, 116.8, 117.2, 117.8,
+
128.9, 132.6, 133.9, 136.1, 148.2, 158.1, 169.9; HRMS (EI, M ) m/z calc. for
12 13 3
C H N O 215.1059, found 215.1055.
3-Acetamido-2-quinolinone (5). To 0.5 mL of acetic anhydride was added
Nꢀacetylglycine (128 mg, 1.1 mmol). The mixture was stirred in a nitrogen
atmosphere at 120 for 10 min. Then, the aldehyde substrate (oꢀ1a, oꢀ1b, or oꢀ1c, 1
℃
mmol) and sodium acetate (123mg, 1.5 mmol) were added into the solution and
stirred under reflux for 18 h. The solution was extracted with ethyl acetate and the
extracted solution was dried with anhydrous sodium sulfate and concentrated by a
rotary evaporator. The crude product was purified by column chromatography with
hexane/ethyl acetate (1:1) as a mobile phase to get 5, and yields were 78, 60 or 66%,
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respectively. HꢀNMR (CDCl
3
3
, 300 MHz): δ 2.29 (s, 3H, CH ), 7.23ꢀ7.32 (m, 2H,
PhH×2), 7.44 (t, 1H, PhH), 7.61 (d, 1H, PhH), 8.46 (bs, 1H, NH), 8.82 (s, 1H, CH),
13
1
1.31 (s(broad), 1H, NH) ; CꢀNMR (CDCl
3
, 75MHz): δ 24.8, 115.3, 120.7, 122.0,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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8
9
0
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7
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1
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9
0
1
2
3
4
5
6
7
8
9
0
+
1
23.5, 127.8, 127.9, 128.6, 133.5, 158.7,169.2; HRMS (FAB, M ) m/z calc. for
11 10 2 2
C H N O , 202.0742, found 202.0742.
2
(1H)-Quinolinone (6). To 0.5 mL of acetic anhydride was added
2
ꢀacetylamidobenzaldehyde (oꢀ1a) (163 mg, 1 mmol) and sodium acetate (123 mg,
1
.5 mmol). The mixture was stirred in a nitrogen atmosphere under reflux for 18 h.
The solution was extracted with ethyl acetate and the extracted solution was dried
with anhydrous sodium sulfate and concentrated by a rotary evaporator. The crude
product was purified by column chromatography with hexane/ethyl acetate (3:1) as a
1
mobile phase to get 6, and yields was 76%. HꢀNMR (CDCl
3
, 300 MHz) δ 6.74 (d,
1H, CH), 7.22 (t, 1H, PhH), 7.43ꢀ7.58 (m, 3H, PhH×3), 7.82 (d, 1H, CH), 12.51
13
(s(broad), 1H, NH) ; CꢀNMR (CDCl
3
, 75MHz) δ 116.2, 119.9, 121.3, 122.7, 127.8,
1
30.7, 138.4, 141.1, 164.6. The data obtained for the products are in agreement with
1
7g
those reported in the literature.
Cis-4-(2-(Dimethylamino)benzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one
o-7). To oꢀ4 (215 mg, 1 mmol) and sodium carbonate (212 mg, 2 mmol) in 3 mL of
(
dried THF was slowly added iodomethane (292 mg, 2 mmol). The mixture was stirred
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in a nitrogen atmosphere at room temperature until the reaction was complete. The
solution was extracted with ethyl acetate and the extracted solution was dried with
anhydrous sodium sulfate and concentrated by a rotary evaporator. The crude product
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was purified by column chromatography with hexane/ethyl acetate (1:1) as a mobile
1
phase to get oꢀ7, and the yield was 55%. HꢀNMR (CDCl
3
, 300MHz) δ 2.38 (s, 3H,
CH
3
), 2.78 (s, 6H, CH
3
), 3.20 (s, 3H, CH
3
), 7.01ꢀ7.07 (m, 2H, PhH), 7.30 (t, J = 7.4
13
Hz, 1H, PhH), 7.56 (s, 1H, CH), 8.44 (d, J = 7.4 Hz, 1H, PhH); CꢀNMR (CDCl
3
,
7
1
5MHz) δ 15.6, 26.6, 45.2, 117.9, 122.1, 125.5, 127.1, 130.7, 133.1, 137.5, 155.1,
+
17 3
61.5, 170.8; HRMS (EI, M ) m/z calc. for C14H N O 243.1372, found 243.1367.
ACKNOWLEDGMENT
We thank National Science Council of Taiwan for financial support
NSC101ꢀ2113ꢀMꢀ006ꢀ001ꢀMY3).
(
Supporting Information. The energy and redundant internal coordinates of oꢀ4, oꢀ7
1
13
and pꢀ7, the H and C NMR spectra of oꢀ3d, oꢀ4, 5, 6, oꢀ7 and pꢀ7, and the
electronic absorption spectra of oꢀ4, oꢀ7 and pꢀ7 in various solvents. This material is
available free of charge via the Internet at http://pubs.acs.org.
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